Therapeutic composition for use in the prevention and treatment of bone metastases

ABSTRACT

The invention is relates to drugs on the basis of antibodies against non-cellular bone matrix proteins, especially BSP, for use in the prevention and treatment of bone metastases. According to the invention, the antibodies are induced in the patient by recombinantly expressing antigenic determinants of bone matrix proteins in  Listeria  and anchoring them on the surfaces thereof. The antigenic determinants are selected according to whether they are characteristic of antigens that are expressed by the tumor cells themselves.

FIELD OF THE INVENTION

The invention relates to medicaments for use in the prevention andtreatment of bone metastases which are based on antibodies againstnon-cellular bone matrix proteins.

BACKGROUND OF THE INVENTION

A primary cancer is often followed by secondary bone tumors. Despite allmedical advances, bone tumors cannot be healed as they generally occurspread-out and can hardly be treated surgically. The probability of bonemetastases is larger than 96% in the case of multiple myeloma, between65% and 75% in the case of cancers of the breast and prostate, andbetween 30% and 50% in the case of cancers of the lung, kidney, cervixand bladder. Systemic chemotherapies have hardly any effect. Initialsuccesses have been achieved using therapeutic antibodies. However, whentherapeutic antibodies are used, there is the problem that they aremostly directed against targets which are only present on proliferatingtumor cells. Generally not detectable are non-proliferating tumor cellsin the blood circulation and latent metastases.

There is a close relationship between the occurrence of the bone matrixproteins osteopontin (OPN), osteonectin (ON) and bone-sialoprotein II(BSP) in the primary tumor on one hand, and the later occurrence ofsecondary blastomas of the bones on the other hand. The cells of anosteotropic metastasizing primary tumor nearly always expresssignificant amounts of BSP or OPN (I. J. Diel et al., Clinic Cancer Res,1999, 5:3914; A. B. Tuck et al., J Mammary Gland Biol Neplasia, 2001,6:419; P. S. Rudland et al., Cancer Res 2002, 62:3417; D. Agrawal etal., J Natl Cancer Inst, 2002, 94:513; D. Waltregny et al., J Bone MinerRes, 2000, 15(5):834; A. Bellahcène et al., J Bone Miner Res, 1996,11:665; D. Waltregny et al., J Nat Cancer Inst, 1998, 90:1000; A.Bellahcène et al., Int. J. Cancer, 1996, 69:350; WO 02/100901(Immundiagnostik AG), WO 02/25285 (Smith et al.)). BSP is aphosphorylated glycoprotein with a relative mass of about 80 kDa inSDS-PAGE. The cDNA for BSP codes for a peptide sequence of about 33 kDa(L. W. Fischer et al., J Biol Chem, 1990, 265:2347; U.S. Pat. No.5,340,934 (Termine)). It represents about 10% to 15% of the non-collagenproteins in the bone matrix, and it is normally expressed by cells thatare involved in the formation of dentine, bone and cartilage, such asosteoblasts, developing osteocytes, hypertrophic chondrocytes,odontoblasts and cementoblasts. As an adhesion molecule, BSP supportsthe attachment and spreading of osteoblasts and osteoclasts on the bonetissue matrix. The switching-off of the BSP-gene in knock-out mice did,however, not lead to a visible disruption of skeleton formation.However, BSP has been attributed a role in bone microcalcification andbone colony formation of tumor cells (V. Castronovo et al., Int J Oncol,1998, 12:308; A. Bellahcène et al., Int J Cancer, 1996, 69:350).

Free BSP is bound with high affinity by complement factor H in bodyfluids. There are many antibodies against BSP peptide structures,recombinant BSP and BSP isolated from bone, which do not recognise andbind BSP in serum (L. W. Fisher et al., Acta Orthop Scand Suppl 1995,266:61; J. T. Stubbs (III) et al. J Bone Miner Res, 1997, 12(8):1210).The 150 kDa large factor H molecule most likely encloses the BSPmolecule in such a way that these antibodies cannot bind. Trophoblastsand BSP producing tumor cells are therefore also protected from anattack by the immune system (N. S. Fedarko et al. J. Biol. Chem., 2000,275, 16666-16672; WO 00/062065). The heavy glycosylation of BSP may alsoplay a role in this observation. Furthermore, BSP may bind through itsRGD sequence (arginine-glycine-aspartic acid) to alpha(v)beta(3)integrin receptors on the cell wall. Thus, BSP is further involved inthe adhesion, dissemination and orientation of the endothelial cells andthe angiogenesis around a tumor (A. Bellahcène et al. Circ Res. 2000,86(8):885-91). These properties make BSP, alongside OPN and ON in thefamily of non-collagen integrin receptor binding glycoproteins, astarting point for medicaments of all kinds (U.S. Pat. No. 5,780,526;U.S. Pat. No. 5,767,071; U.S. Pat. No. 5,792,745; U.S. Pat. No.5,989,383; U.S. Pat. No. 5,773,412; U.S. Pat. No. 5,849,865).

There have been attempts to inhibit through RGD-antagonists the bindingof BSP to the vitronectin and integrin receptors of the tumor andendothelial cells (U.S. Pat. No. 6,069,158; U.S. Pat. No. 6,008,213;U.S. Pat. No. 5,849,865; van der Pluijm et al., Cancer Res., 1996, 56,1948-1955). EP 1 084 719 (DePuy Orthopaedics Inc.) describes BSP anactive agent for supporting the repair of damaged bone and connectivetissues. WO 94/11310 (Alfa-Laval Agriculture Intern. AB) discloses aBSP-binding protein from Staphylococcus aureus for a treatment ofinfections and inflammatory diseases of the bone. WO 02/100899(Armbruster Biotechnology GmbH) discloses an active ingredient againstbone metastases based on antibodies against BSP. WO 00/36919 (Univ.Virginia Patent Found.) describes regulatory elements for control andinhibition of BSP expression in tumor cells and connective tissue cells.Finally, EP 0 020 789 (DKFZ) discloses an inhibition of cell migrationand bone metastasis formation by antisense-oligonucleotides (quod videAdwan-Hassan et al. in Cancer Gene Therapy, 2003:1; Intern J Oncol,2004, 24:1235-1244; Proc Am Assoc Cancer Res Ann, 2003, 44:56). WO2006/036550 (Trustees of the Univ. of Pennsylvania; published after thepriority date of this application) further describes vaccines on thebasis of Listerium and fusion proteins of listeriolysin and CD8⁺-T-cellepitopes (Her-2) for a treatment of osteotropic tumors and carcinoma.

Therefore, it has been examined what causes a primary tumor, which cannormally be surgically removed, to produce metastases and how bonemetastases may be prevented and what is needed for their treatment oreventual cure. In previous attempts, it proved disadvantageous that atherapy based on antisense oligonucleotides or antibodies, wheneffective at all, can only be maintained effective for a limited period.There is not only a problem of dose and application, but also due to thedevelopment of autoantibodies against the therapeutic immunoglobulinsand regulatory nucleotides. One should further not forget that the bodycontains natural endogenous BSP so that an immune reaction againstendogenous BSP is inhibited. On the other hand, prophylaxis or a directtreatment of bone metastases must be carried on over very long periodsin order to be potentially successful. The danger of an occurrence ofbone metastases will last for decades after a treatment of a primaryosteotropic tumor. Thus, there is a need for a therapeutic compositionthat sustainably prevents a colony forming and development of bonetumors, and fights any existing bone metastases.

SUMMARY OF THE INVENTION

The problem is solved by a therapeutic composition in accordance withclaim 1. Preferred embodiments of the invention are described in thedependent claims.

According to the invention, the pharmaceutical composition for treatmentand prophylaxis of bone tumors and metastases that preferably colonizeinto bone tissue contains as active ingredient dead or weakly pathogenicmicro-organisms, which contain a gene for antigenic fragments of thebone sialoprotein and express one or more bone sialoprotein antigensthat differ in at least one structural feature from endogenous bonesialoprotein of normal osteoblasts so that their administration producesan immune reaction against the altered bone sialoprotein. The expressedbone sialoprotein molecules preferably possess structural features of abone sialoprotein that is specifically expressed by the osteotropiccells of a primary tumor. According to the invention, the microorganismis selected from bacteria, viruses and monads, preferably fromGram-positive bacteria such as Listeria. It may also be selected fromthe species Aeromonas, Bartonella, Bruceila, Bacilli, Bacillus subtilis,Lactobacilli, Pseudomonades, Staphylococci, Yersinia, Campylobacter,Clostridia, Enterobacteriaceae, Legionella, preferably Listeria, morepreferably Listeria monocytogenes, Mycobacterium, Rhenibacterium,Rhodococcus, bacteria of the species Yersinia, Escherichia, Shigella,Salmonella, and bacteria, which may survive in a eukaryotic hostorganism. Particularly preferred is an embodiment where themicroorganism carries one or more BSP antigen determinants anchored toits surface, more particularly, tumor-typical bone sialoprotein orfragments thereof. The microorganism may carry anchored to its surfacean underglycosylated bone sialoprotein antigen or fragments thereof. Forhigh therapeutic activity, it is important that the bone sialoproteinantigen possesses an epitope, which, when in a complex of BSP andcomplement factor H, is free for the binding of an antibody. Accordingto the invention, such a bone sialoprotein antigen contains one or morecopies of the following amino acid sequences:

YTGLAAIQLPKKAGD SEQ. ID NO. 5 TGLAA SEQ. ID NO. 3 YTGLAA SEQ. ID NO. 4YESENGEPRGDNYRAYED SEQ. ID NO. 6 LKRFPVQGG SEQ. ID NO. 7EDATPGTGYTGLAAIQLPKKAG SEQ. ID NO. 10

In one embodiment of the invention, the pharmaceutical compositioncomprises as an active ingredient an immunogen with a hapten, which ispresent on bone sialoprotein from tumor cells, and more preferably theantigen determinant of bone sialoprotein in at least two or more copies.The pharmaceutical composition of the invention can be used for atreatment of tumors selected from the group comprising tumors of theprostate, breast, lung, kidney and thyroid, tumors of the circulatorysystem, lymphoid system, cardio-vascular system, neurological system,respiratory tract, intestinal tract, endocrine system, skin includingadnexa, musculoskeletal system and urogenital system, including thekidneys.

A further aspect of the invention relates to a method for developing atherapeutically active composition comprising the of steps: (i)selecting a protein relevant for a disease; (ii) cloning and expressionof an antigenic structure of the relevant protein in a microorganism,which expresses, secretes and presents an antigenic fragment thereof,anchored to the membrane, on its cell surface; (iii) eliciting ofantibodies against the antigenic fragment of the disease-relevantprotein; (iv) testing of the antibodies for therapeutic activity.Preferably, the microorganism is a Gram-positive bacterium, morepreferably, the microorganism is Listerium. In the process, sera ofmammals are used to screen the antigens, and the sera are then examinedfor the presence antibodies against the antigenic fragment. For thepurpose of this invention, disease-relevant proteins are examined, whichhave a physiological function in the colonialization of tumor cells intobones, for example, the extracellular bone matrix proteins bonesialoprotein (BSP), osteopontin (OPN), osteonectin (ON) and growthfactors for osteotropic tumors. The invention also incorporatestherapeutically useful antibodies and vaccines obtained by this process.

Another aspect of the invention concerns a process for treatment andprevention of bone tumors and metastases, which preferably settle intobone tissue, including the administration of dead or weakly pathogenicmicroorganisms, which possess one or more antigens of the bonesialoprotein anchored on the surface, which differ in at least onestructural feature from endogenous bone sialoprotein of normalosteoblasts, so that their administration elicits an immune reaction,which is directed against the tumor and its disseminating tumor cells.In an alternative process comprising the administration of peptidicmolecules or carrier proteins with antigenic determinants of the bonesialoprotein, which are characteristic for a bone sialoprotein producedby tumor cells, so that an immune reaction is elicited against the tumorand its disseminating tumor cells. A further embodiment of the processcomprises the administration of peptidic molecules or carrier proteinswith antigenic determinants of bone sialoprotein, which arecharacteristic for a bone sialoprotein from tumor cells so that animmune reaction is elicited which acts against the tumor and itsspreading tumor cells. It is preferred in this connection when theantigenic determinants occur several times on the peptidic molecules orcarrier proteins or when the peptidic molecules are coupled tobeta-alanine.

In accordance with the invention, the therapeutic composition fortreatment and prophylaxis of bone tumors and metastases which preferablysettle into bone tissue contains as active agent dead or weaklypathogenic microorganisms, which contain a gene for BSP leading to anexpression of one or more BSP molecules, which differ in at least onestructural feature from endogenous BSP of normal osteoblasts, so that,when administered, an immune reaction against the modified BSP isinduced. It is preferred that the expressed BSP molecules possessstructural features characteristic for the tumor form, such as can befound with BSP from the osteotropic cells of a primary tumor. Therespective microorganism is selected from bacteria, viruses and monads,and is preferably a Gram-positive bacterium, most preferably Listerium.The microorganism may be selected from bacteria propagatingintracellularly in host cells, for example from the species Aeromonas,Bartonella, Bruceila, Bacilli, Bacillus subtilis, Lactobacilli,Pseudomonades, Staphylococci, Campylobacter, Clostridia,Enterobacteriaceae, Legionella, Listeria, Mycobacterium, Rhenibacterium,Rhodococcus, Yersinia, Escherichia, Shigella, Salmonella, and bacteria,which may survive in an eukaryotic host organism, such as Listeria.Bacteria normally not propagating intracellularly may further beimplemented through genetic manipulations with factors that allow themto access cells. Advantageously, the genetically manipulatedmicroorganism contains an exogenous or heterologous suicide gene and canproduce a targeted somatic transgenic modification in the host cells.Particularly preferred is the use of Listeria for the production of atherapeutic composition in accordance with the invention.

A further aspect of the invention concerns a therapeutic composition,wherein the genetically modified microorganism, for example Listerium,has anchored on its surface a BSP antigen, respectively, an antigenicdeterminant of BSP, preferably originating from human BSP and fragmentsthereof, most preferably from glycosylation-deficient BSP and fragmentsthereof. The expressed BSP fragments will then be recognized as foreignin a mammal, particular, when located on the membrane of a microorganismand on the cell surface of infected host cells, respectively. The soelicited autoantibodies then bind to the BSP antigen of osteotropictumor cells. An analogous effect may be achieved by coupling amino acidscharacteristic for bacteria such as beta-alanine (3-aminopropionic acid)with the peptidic antigenic determinants of BSP at the C-terminal end orthe N-terminal end of the peptide or both. An immunogenic BSP and,respectively, a tumor BSP isoform determinant may be produced herebywhich may be used as a vaccine against BSP.

Preferred DNAs for producing specific anti-tumor-BSP antibodies encodeamongst others the following sequences of human bone sialoprotein(SWISSPROT: SIAL_HUMAN, Acc. No. P21815) and its homologues:

SEQ ID NO: 1 X-YTGLAAIQLPKKAGD-Z SEQ ID NO: 2X-FSMKNLHRRVKIEDSEENGVFKYRPRYYLYKHAYFYPHLKRFPVQGSSDSSEENGDDSSEEEEEEEETSNEGENNEESNEDEDSEAENTTLSATTLGYGEDATPGTGYTGLAAIQLPKKAGDITNKATKEKESDEEEEEEEEGNENEESEAEVDENEQGINGTSTNSTEAENGNGSSGVDNGEEGEEESVTGANAEGTTETGGQGKGTSKTTTSPNGGFEPTTPPQVYRTTSPPFGKTTTVEYEGEYEYTYDNGYEIYESENGEPRGDNYRAYEGEYSYFKGQGYDGYDGQNYYH HQ-Z

The highlighted threonine is not or incompletely or differentlyglycosylated in the BSP tumor isoform. In one embodiment, this threonineis converted into an amino acid which can not be glycosylated. X and Zrepresent amino acid residues and/or peptide moieties, for example, amembrane anchor, poly(histidine), poly(His)₅₋₁₂, or beta-alanine. SEQ IDNO: 2 may be modified as follows: Position 179 Gly→Val; Position 252Val→Ala, Position 254 Glu→Asp; Position 279 Asp→Gly.

In one embodiment of the invention the therapeutic composition bringsabout the formation of endogenous antibodies against a BSP, whichposttranslational glycosylation is modified or incomplete in the regionof amino acids 120 to 135 (SWISSPROT: SIAL_HUMAN, Acc. No. P21815)compared to regular BSP from bones.

Preferred is the induction of endogenous autoantibodies which recognizea hBSP-epitope comprising the amino acid sequence TGLAA (SEQ ID NO: 3)or YTGLAA (SEQ ID NO: 4), and optionally sugar groups and a carriermolecule.

Hence, the vaccine of the invention gives rise to endogenousautoantibodies against a BSP tumor isoform. The so induced immunitytherefore protects against a docking of metastasizing osteotropic tumorcells to bone tissue and results in a cell-mediated cytotoxicity againstcells producing the tumor isoform of BSP.

Another aspect of the invention concerns a composition, wherein the BSPantigen contains an antigenic determinant, respectively an epitope,which is free for the binding of an antibody even when in a complex ofcomplement factor H and BSP. The antigenic determinant of BSP maycontain one or more copies of the following amino acid sequences:

TGLAA SEQ ID NO: 3 YTGLAA SEQ ID NO: 4 YTGLAAIQLPKKAGD SEQ ID NO: 5YESENGEPRGDNYRAYED SEQ ID NO: 6 LKRFPVQGG SEQ ID NO: 7

A preferred goal is hereby a therapeutic composition wherein the activeingredient possesses a hapten that is also present on BSP from tumorcells. The composition of the invention can be used for a treatment oftumors and carcinoma selected from a group comprising tumors of theprostate, breast, lung, kidneys and thyroid, tumors of the circulatorysystem, lymphoid system, cardiovascular system, neurological system,respiratory tract, digestive tract, endocrinal system, skin includingadnexa, musculoskeletal system and urogenital system, including thekidneys.

A further aspect of the invention relates to a process for thedevelopment of an active composition, or of a vaccine, comprising thefollowing steps: (i) selecting a protein relevant for the disease; (ii)cloning and expression of an antigenic structure of the relevant proteinin a microorganism, which expresses, secretes and presents on the cellsurface, anchored to the cell membrane, the antigenic structure; (iii)eliciting of antibodies against the antigenic structure of thedisease-relevant protein; (iv) testing of the antibodies for therapeuticactivity and use of the antibodies in a therapeutic composition, forexample in a vaccine. The microorganism that presents the antigenicstructure on its surface may be a Gram-positive bacterium, preferablyListerium. Preferred microorganisms induce a somatic transgenicity inhost cells and they present the antigenic structure, inclusiveposttranslational modifications, on the surface of the host cells.Particularly preferred are microorganisms such as Listerium, which areable to break the immunotolerance against an antigen expressing tumor.This may be achieved in particular by anchoring and presenting theantigen on the surface of the microorganism, or by coupling the antigenwith bacteria characteristic amino acids such as beta-alanine. Themicroorganism may be inactivated prior to step (iii). It is furthercontemplated to test sera of mammalians for the presence of antibodiesagainst the antigenic structure of the disease-relevant protein, and inparticular to examine the proteins which have a physiological functionin the settling of tumor cells in bones. The most promising proteins inthis connection are mainly the extracellular bone matrix proteins bonesialoprotein (BSP), osteopontin (OPN), osteonectin (ON) and the growthfactors for tumors. The process of the invention may be broadly appliedas a screening process. In this case a multitude of antigenic structuresof disease-relevant proteins are cloned in microorganisms, expressed andanchored on the surface, and mammalian sera screened for antibodiesagainst the antigenic fragments, in order to select for therapeuticallyuseful antigenic determinants, fragments and haptens, as well asantibodies. The anamnesis of these mammals, respectively patients,especially of the ones with spontaneous recovery, then points to apossible activity of the antibodies. This results in therapeuticallyactive antibodies and microorganisms having the antigenic structure onthe surface, and finally vaccines on basis thereof.

Further aspects and advantages of the invention are described in thedetailed description of the invention and by attached figures andexamples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a block diagram of the process of the invention for theproduction of bacteria having antigen determinants from BSP, whichelicit an immune reaction against osteotropic tumor cells and thesettling of tumor cells in bones;

FIG. 2 shows a representation of generated BSP fragments for recombinantexpression of BSP antigen segments on carrier bacteria; RGD:cell-binding motif; YXY: tyrosine-rich region; E8: glutamic acid-richregion; T-Epitop: BSP tumor epitope (BSP-position 125-130);

FIG. 3A shows a representation of the proportion of positive bacteriawhich after FACS analysis have on their surface functionally anchoredBSP fragments (vBSP-1, -2, -3, -4 or eBSP), as evidenced by atumor-epitope specific polyclonal antiserum (rabbit)—carrier bacteriawith no BSP on the surface were used as negative control;

FIG. 3B-3G show representations of the proportion of positive bacteria,which after FACS analysis carry anchored vBSP-1, -2, -3, -4 and eBSP forvarious secretion signals (pIEX-A, -B, and -C), vectors (pIEX, pIUS),with a carrier protein (pXC-Add, PS-Add), as evidenced by monoclonalantibodies against the myc-tag—carrier bacteria with no recombinantprotein on their surface were used as a negative control;

FIG. 4 shows diagrams of a flow-through cytometry of carrier bacteriafor functionally anchored BSP fragment on the surface priorinactivation—A) anchoring of vBSP-3 on carrier bacteria grown in BHIculture medium; B) anchoring of vBSP-3 on carrier bacteria grown inminimal medium, and the fluorescence activated cell sorting (FACS) beingcarried out with polyclonal rabbit anti-tumor eBSP antibodies andcarrier bacteria without BSP determinants on the surface as control;

FIG. 5 shows diagrams of the flow-through cytometry of carrier bacteriain accordance with FIG. 4 post inactivation—A) anchoring of vBSP-3 whengrown in BHI-medium; B) anchoring of vBSP-3 when grown in minimalmedium;

FIG. 6 shows diagrams of the flow-through cytometry of carrier bacteriaprior and post inactivation for functionally anchored BSP fragment vBSP2or vBSP 4—anchoring of vBSP2 when carrier bacteria were grown in BHIculture medium A) prior inactivation and C) post inactivation; anchoringof vBSP4 when carrier bacteria were grown in BHI medium B) priorinactivation and D) post inactivation;

FIG. 7 shows a flow-through cytometry of carrier bacteria forfunctionally anchored tumor eBSP epitope, fused with a carrier protein,when detected A) with a monoclonal antibody against the carrier protein,B) with polyclonal tumor-epitope specific rabbit antibodies, and carrierbacteria without any recombinant protein on their surface as control—C)schematic representation of the expression cassette of the eBSPconstruct, SS: signal sequence, S-Tag: immune tag, myc-Tag: immune tag;Anker: anchor sequence;

FIG. 8 shows a representation of the expression cassette of theanchoring constructs pIUSInd-mBSPIx-6x: pInd: Promotor; SS: signalsequence; S-Tag: immunological tag; SP: Spacer (SGGGGSA)—SEQ ID NO: 8;myc-Tag: immune tag; Anker: anchor sequence;

FIG. 9 shows a flow-through cytometry of carrier bacteria with variouscopy numbers of BSP tumor epitopes when grown in BHI-medium—functionallyanchored BSP tumor epitope on the surface was detected withtumor-epitope specific rabbit antibodies, and carrier bacteria withoutBSP immune tags on the surface (pERL4-LLO) were used as control:pIUS-eBSP anchors only one and pIUS-mBSP-1x-6x anchors multimers (1-6)of the BSP tumor epitope on the surface of the carrier bacteria;

FIG. 10 shows the diagram of a flow-through cytometry of carrierbacteria grown in minimal medium for surface-anchored functional BSPtumor epitopes in various numbers, detected with a tumor eBSP antiserumfrom rabbit, and carrier bacteria without BSP immune tags on the surface(pERL4-LLO) as control—pIUS-mBSP-1x-6x: anchoring of a BSP tumor epitopeas a multimer (1x-6x) on carrier bacteria;

FIG. 11 shows the diagram of a flow-through cytometry of carrierbacteria post inactivation for surface-anchored functional BSP tumorepitopes in various numbers, when detected with a rabbit tumor eBSPantiserum, and carrier bacteria without BSP immune tags on the surface(pERL4-LLO)—A) pIUS-mBSP-1x-6x: anchored constructs, which have the BSPtumor epitope anchored as a multimer (1x-6x) on carrier bacteria asshown in FIG. 8; B) FACS-diagram of inactivated carrier bacteria grownin minimal medium, which carry the BSP tumor epitope as a 5-multimeranchored to the surface; C) FACS diagram of inactivated carrier bacteriagrown in BHI-medium, which carry the BSP tumor epitope as a 6-multimeranchored to the surface.

DETAILED DESCRIPTION OF THE INVENTION

Osteotropic tumors of the prostate, breast, lung, kidney and thyroiddiffer from less malign tumors inter alia by the fact that they containBSP expressing cells and can disseminate into the bone tissue. Eventhough the process of metastasis into bones is complex and notunderstood, the presence of tumorgenic BSP in serum allows for a safeprognosis of bone metastases. The presence of tumorgenic BSP in the bonematrix may further be used as a rating of the bone remodeling by bonemetastases. BSP from tumor cells possesses other posttranslationalmodifications compared to endogenous BSP of normal osteoblasts. Thus,IgY-antibodies could be produced in chicken which specifically recognizea BSP secreted from cells of osteotropic tumors, called hereintumor-eBSP. In animal experiments, these antibodies are strikinglyactive against induced bone metastases of human cancer cells (WO02/100899). The therapeutic composition of the invention for treatmentand prevention of bone tumors elicits in the patient endogenousantibodies against BSP and in particular against tumor specific eBSP.Presumably, the so induced autoantibodies against tumorgenic eBSPinterfere with the docking mechanism of the osteotropic cells from theprimary tumor. Since BSP is bound and masked by factor H of thecomplement system, many eBSP expressing secondary cells of the primarytumor can evade from being attacked by the immune system and necrosis bythe complement system. Also the bringing about of apoptosis would bevery important. The death of autoreactive lymphocytes in the thyroidgland usually ensures that the immune system does not attack endogenousantigens. The therapeutic composition of the invention overcomes thesefalse self-protection mechanisms, because, inter alia, an immunereaction against tumorgenic BSP from tumor cells is specificallyinduced, and the antibodies bind to an epitope of BSP, which remainsfree and accessible even when enshrouded by complement factor H fromserum. The generated cell-mediated cytotoxicity causes a necrotic orapoptotic death of the target cells. Contrary to conventionaltherapeutic approaches, no tumor BSP specific antibodies areadministered but a cell-mediated immunity produced in the patient by acombined xeno- and idio-immunization against a protein specific fortumors metastasizing into bones and presumably required for theirdissemination into bones.

The therapeutic composition may contain dead or weakly pathogenicmicroorganisms, which contain a DNA sequence encoding BSP or a fragmentthereof integrated in an episomal vector. Consequently, the therapeuticcomposition corresponds to a vaccine, or a live vaccine, in which aforeign BSP-encoding DNA is translated and expressed as BSP antigens andtumor-eBSP antigens, respectively, so that the patient developsantibodies against presented foreign BSP and tumor eBSP antigens. The soinduced auto-immunity against tumor BSP presumably protects against thedocking of metastasizing osteotropic cells of the primary tumor to thebone tissue and leads to a cell-mediated cytotoxicity against tumor BSPproducing tumor cells. Both mechanisms evolve into a prophylactic and atherapeutic activity against bone metastases.

Further, the activity of the therapeutic composition may be enhanced andmodulated by the addition of antibodies, especially anti-tumor BSPantibodies, ligands, especially RGD binding ligands, inhibitors whichinteract with adhesion molecules, membrane associated proteases orreceptors mediating chemotaxis, for example chemokine receptors, as wellas apoptosis inducing substances such as antibodies or proteins andpeptides obtained from natural and artificial peptide libraries.Peptides from BSP and eBSP, which were made immunogenic by coupling withbeta-alanine, appear to be especially promising.

Especially preferred is a therapeutic composition, in which thedeactivated or weakly pathogenic micro-organisms lead to the expressionof tumor eBSP in a form where the tumor eBSP is bound to a plasmamembrane. The plasma membrane may be the plasma membrane of themicro-organism or the cell membranes of host cells of the patient, inwhich the genetic information of the tumor BSP had been specificallyintroduced.

Furthermore, it is useful to modify the introduced BSP-DNA in such a waythat after expression, molecules similar to tumor BSP are obtained. Thepossible locations for a modification are especially on those positionson the DNA, which code amino acids, which are posttranslationally N orO-glycosylated. Hence posttranslational modifications may be removedthrough targeted point mutations. Namely, tumor BSP differs fromendogenous BSP especially by a modified or incomplete posttranslationalglycosylation. In order to increase the antigenicity of the cloned andexpressed fragment, it may be advisable to couple several repeatsthereof in line.

The amino acid sequence of human BSP contains four potentialN-glycosylation sites at positions 88 (NTT), 161 (NGT), 166 (NST) and174 (NGS). No consensus sequence is known with respect toO-glycosylation sites. All identified N-glycan structures can be foundon BSP isolated from bones as well as on BSP from tumor cells. There aredifferences however in the percentage of the respective structureswithin the total amount of N-glycans. The major amount of N-glycans onBSP from bones consists of triantennary structures (58%) whereas forexample they consist in the degenerate EBNA cell line of tetraantennarystructures (48%). Furthermore, the human BSP molecule has at least eightO-glycosylation sites, five on the peptide 211-229 (TTTSP . . . QVYR)and three at most on the peptide between amino acid 120 and amino acid135 with the sequence TGLAA (SEQ ID NO: 3). Of these, the threonines inthe sequence DATPGPT (SEQ ID NO: 9) are O-glycosylated on recombinantlyexpressed BSP from EBNA cells. A third O-glycosylation can be found onBSP isolated from bones. No third glycosylation location is present onrecombinant BSP. This glycosylation site is presumably located on theTGLAA-BSP partial structure (SEQ ID NO: 3).

Because of the advantageous results obtained with antibodies againstthis partial structure of human BSP, the respective DNA sequence,coupled to a DNA encoding a carrier peptide and a membrane anchor(poly-His, Internalin-A sequences), was introduced as foreign DNA intothe micro-organism and expressed—either in the micro-organism itself, orin the somatic transgenic host cells of the patient. The expressed tumorBSP fragments were recognized as foreign in mammals, because they werelocated on the membrane of a micro-organism, or on the surface ofinfected host cells. The so induced own antibodies bind to the BSP ofthe osteotropic tumor cells.

As said before, the preferred BSP peptide fragments are

SEQ ID NO: 1 X-YTGLAAIQLPKKAGD-Z SEQ ID NO: 2X-FSMKNLHRRVKIEDSEENGVFKYRPRYYLYKHAYFYPHLKRFPVQGSSDSSEENGDDSSEEEEEEEETSNEGENNEESNEDEDSEAENTTLSATTLGYGEDATPGTGYTGLAAIQLPKKAGDITNKATKEKESDEEEEEEEEGNENEESEAEVDENEQGINGTSTNSTEAENGNGSSGVDNGEEGEEESVTGANAEGTTETGGQGKGTSKTTTSPNGGFEPTTPPQVYRTTSPPFGKTTTVEYEGEYEYTYDNGYEIYESENGEPRGDNYRAYEGEYSYFKGQGYDGYDGQNYYH HQ-Zwherein the highlighted threonine is not or incompletely glycosylated inBSP from tumor cell, or in a another way. SEQ ID NO: 2 may be modifiedas follows: position 179 Gly→Val; position 252 Val→Ala; position 254Glu→Asp; Position 279 Asp→Gly.

In one embodiment of the invention the therapeutic composition inducesthe formation of endogenous antibodies against a BSP, whichposttranslational glycosylation in the region of amino acids 120 to 135(SWISSPROT: SIAL_HUMAN, Acc. No. P21815) is modified or incompletecompared to normal BSP from bones. Preferred is the induction ofendogenous antibodies that recognize a hBSP epitope, which includes theamino acid sequence TGLAA (SEQ ID NO: 3) or YTGLAA (SEQ. ID NO: 4) andoptionally sugar groups as well as a carrier molecule.

The vaccine of the present invention therefore results in endogenousantibodies against BSP from tumor cells. The so induced immunitytherefore protects against the docking of metastasizing osteotropictumor cells into bone tissue and assists in the development of acell-mediated cytotoxicity against tumorgenic BSP producing cells.

The pharmaceutical composition in accordance with the present inventionis especially useful in the treatment of tumors from the groupcomprising tumors of the prostate, breast, lung, kidney, thyroid,circulatory system, lymphoid system, cardiovascular system, neurologicalsystem, respiratory tract, digestive tract, endocrine system, skinincluding adnexa, musculoskeletal system and urogenital system.

A further aspect of the invention relates to a process for thedevelopment of vaccines, especially against tumors in general andosteotropic tumors, which metastasize into bones. This process comprisesthe following steps: (i) identification of a protein relevant for thecondition such as e.g. BSP; (ii) formation of carrier organisms with oneor more chosen regions (immune tags) of the protein relevant for thecondition, which express and carry on the cell surface, anchored to thecell membrane, the immune tag; (iii) testing of preferably inactivecarrier organisms, for whether they induce an immune reaction againstthe immune tags of the protein relevant for the condition; (iv)examination of a number of subjects, including sick and healthypatients, for antibodies against said immune tags of the proteinrelevant for the condition, for example by a screening of blood sera and(v) selecting carrier organisms with immune tags on the surface asvaccine bacteria in accordance with the finding as to which subjects andwith which anamnesis, including recovered healthy patients, possessantibodies that bind to one or more immune tags of said protein relevantfor the condition. As steps (i) to (iii) are relatively fast and mostimportantly can be carried out in parallel, and as blood sera for use instep (iv) and corresponding health records (v) are available by themillion, useful vaccine bacteria with anchored immune tags can beproduced and identified by a statistical analysis of the health records.The occurrence of natural antibodies against one or more immune tags ofsaid protein relevant for the condition in any blood serum is already anindication per se that the one or more selected immune tags aremedically relevant. The relevance may be determined by comparing thecorresponding anamneses. As step (iv) can done by analytical machinesfor numerous and abundantly available blood sera, relevant andnon-relevant antigen fragments can readily be identified by statisticalanalysis without the need for a medical anamnesis. In particular bloodsera of patients with spontaneous recovery or a very mild course ofdisease should be tested in the search for tumor relevant immune tags.

EXAMPLES Example 1 Expression Vectors and Micro-Organisms forImmunization

The cDNA of human BSP (SEQ. ID NO: 2), respectively the below mentionedBSP fragments were cloned in the polycloning site of an episomal pSOGshuttle vector (Machner et al., J. Biol. Chem., 2001, 276 (43), 40096;L. W. Fisher et al., J. Biol. Chem., 1990, 265(4), 2347-51). In order toachieve an anchoring of the cloned heterologous BSP antigens on thesurface of the Listeria, an expression and anchoring cassette was builtinto the E. coli/Listeria shuttle vector (erythromycin resistance, ColE1origin of replication, gram-positive minimal replicon for gram-positivebacteria). All components of the expression and anchoring cassette wereamplified using PCR from the respective organisms and cloned into theshuttle vector. The relative order of the individual components of theexpression and anchoring cassette is shown in FIG. 8.

The promotor (pind) of the Listeriolysin gene from Listeriamonocytogenes was used for the expression of recombinant peptides(Mengaud et al. Infect. Immun. 1989, 57:3695). The promotor was clonedtogether with the 5′-untranslated region of the Lysteriolysin gene(Accession No. NC003210) upstream of a nucleotide sequence encoding thesignal peptide (SS) of Listeriolysin (LLO—protein sequence inSwiss-Prot: Q724L1; nucleotide sequence—Genbank Accession Nos. P13128,X15127). The identification of the signal sequences was done inaccordance with the literature and using the software SignalP vs. 2.0(Nielsen et al., Protein Engineering, 1999, 12:3). The signal peptide,which is usually about 25 amino acids long, transports the recombinantpeptides across the cell membrane of the carrier bacterium and is thensplit off. In one embodiment were upstream and downstream of the genesencoding the BSP epitopes further nucleotide sequences introduced whichencoded two immune tags (S-tag of Novagen, Darmstadt; upstream, myc-tagof NanuTec GmbH, Frankfurt; downstream). When expressed the S-tag andmyc-tag facilitate the immunological characterization of the anchoringof the BSP epitopes on the surface of the Listeria. In order to achievea secure anchoring of the recombinant heterologous antigen determinanton Listeria, a sequence from Internalin A (InlA) of L. monocytogenes wasintegrated downstream of the myc-tag. The introduced anchor included thepositions 677-800 of the InlA (Swiss-Prot: Q723K6).

Between the two immune tags were then cloned two nucleotide sequenceswhich encoded the BSP tumor epitopes as monomer or as polymer comprisingtwo to six copies of the BSP tumor epitope. The BSP tumor epitope hasthe amino acid sequence -EDATPGTGYTGLAAIQLPKKAG- (eBSP) (SEQ ID NO: 10).The different tumor epitopes were linked via a short spacer with theamino acid sequence -SGGGGSA- (Sp) (SEQ ID NO: 8). The tumor epitope andthe spacer were amplified by a PCR and cloned into the anchoring plasmidpIUSind using standard methods.

Hence, the constructs in the example of pIUSind-mBSP1x-6x in Listerialed to the expression of fusion peptides, which consisted of anN-terminal signal peptide, an S-tag, a (pIUSind-mBSP1x) or several BSPepitopes (pIUSind-mBSP2x-6x), an myc-tag and an anchoring structure. Thesignal peptide effected that the fusion peptide got translocated out ofthe carrier bacterium while the anchoring structure of the Internalin-Aprovided for a “covalent” anchoring of the BSP epitope to the cell wallof the bacterium. The correct reading frame was verified by DNAsequencing and expression; the fusion peptide was recognized byanti-hBSP-antibodies and also by therapeutic IgY antibodies. TheListeria pIUSind-mBSP5x (DSM 18306) and Listeria pIEx-A-vBSP3 (DSM18305), which contain the vectors, were deposited with the DSMZ (GermanNational Resource Centre for Biological Material, Braunschweig).

Example 2 Therapeutically Active BSP Antigen Structures

The epitopes of therapeutically active anti-BSP-IgG and -IgY were mappedto characterize more closely the antigens, respectively haptens, towhich they bind in the immunization experiments against tumorgenic BSP.

TABLE 1 BSP epitopes of therapeutically active anti-BSP-IgG and -IgYglobulins Reaction Position of the strength structural Amino acidchicken - rabbit fragment in BSP sequence IgY IgG (Position, includingLeader) 112-123 - SEQ ID LeuGlyTyrGlyGluAsp − ? NO: 11AlaThrProGlyThrGly 216-227 - SEQ ID GluThrGlyGlyGlnGly − ? NO: 12LysGlyThrSerLysThr 300-311 - SEQ ID PheLysGlyGlnGlyTyr − ? NO: 13AspGlyTyrAspGlyGln 130-144 - SEQ ID IleGlnLeuProLysLys +/− + NO: 14AlaGlyAspIleThrAsn LysAlaThr 124-138 - SEQ ID TyrThrGlyLeuAlaAla − ++NO: 01 IleGlnLeuProLysLys AlaGlyAsp 137-151 - SEQ ID GlyAspIleThrAsnLys− + NO: 15 AlaThrLysGluLysGlu LysGluSerAspGlu 280-317 - SEQ IDSerGluAsnGlyGluPro ++ + NO: 16 ArgGlyAspAsnTyrArg AlaTyrGluAspGluTyrSerTyrPheLysGlyGln GlyTyrAspGlyTyrAsp GlyGlnAsnTyrTyrHis HisGln Humanbone BSP +++ +++

The results show that the known chicken antibodies preferably bind tothe terminal sequence of the BSP whereas the rabbit antibodies bind overa larger range. Therapeutically relevant regions of the human BSP aretherefore:

SEQ ID NO: 1 TyrThrGlyLeuAlaAlaIleGlnLeuPro (positions 124-138)LysLysAlaGlyAsp SEQ ID NO: 3 ThrGlyLeuAlaAla (positions 125-130) SEQ IDNO: 4 TyrThrGlyLeuAlaAla (positions 124-130) SEQ ID NO: 6TyrGluSerGluAsnGlyGluProArgGly (positions 278-295)AspAsnTyrArgAlaTyrGluAsp SEQ ID NO: 7 LeuLysArgPheProValGlnGlyGly(N-Terminus)

For a secondary delimitation of the tumor relevant BSP structures, thefollowing larger fragments of human BSP were cloned and expressed inbacteria:

-   vBSP-1: 301 amino acids of the hBSP sequence between positions 17    and 318, which represent the full sequence of human BSP (without the    signal sequence).-   vBSP-2: 200 amino acids between positions 57 and 257—vBSP-2 contains    no tyrosine-rich regions and no RGD sequence. vBSP-2 starts    immediately after the first tyrosine-rich region and finishes    immediately before the second tyrosine-rich region.-   vBSP-3: 234 amino acids between positions 84 and 318. vBSP-3 does    not contain the first glutamic acid rich region and extends until    the C-terminal of the BSP.-   vBSP-4: 174 amino acids between positions 84 and 257. vBSP-4 has    been shortened by the first glutamic acid rich region and all    tyrosine-rich regions.-   eBSP: 22 amino acids (-EDATPGTGYTGLAAIQLPKKAG- (eBSP)—SEQ ID NO: 10)    between positions 115 and 137, including one antigenic determinant    of BSP which has been identified as tumor epitope.

FIG. 2 shows the position of the above-mentioned fragments in the BSPprotein in relation to the other relevant structures.

Example 3* Generation of the BSP Constructs Needed for the Anchoring

The DNA sequences of the BSP fragments vBSP-1, -2, -3, -4 and eBSP(Example 2, FIG. 2) were amplified using PCR and cloned into the abovedescribed shuttle vectors. The cloning of the genes was verified bysequencing and then the genes sub-cloned into various secretion andanchoring vectors. The BSP fragments vBSP-1, -2, -3 and -4 were fused atthe C-terminus with an immune tag (myc-tag) to facilitate the detectionof anchored fragments. The only 22 amino acid long fragment containingeBSP was fused to the N-terminus of a bacterial carrier protein to avoidthat the eBSP because of its small size does not protrude from thebacterial cell wall or is masked by other molecules in the cell wall.The clones detailed in table 2 were generated:

TABLE 2 anchoring construct anchoring vector BSP fragment carrierprotein plEX-A-vBSP1 plEX-A vBSP-1 no plEX-A-vBSP2 plEX-A vBSP-2 noplEX-A-vBSP3 plEX-A vBSP-3 no plEX-A-vBSP4 plEX-A vBSP-4 no plEX-B-vBSP1plEX-B vBSP-1 no plEX-B-vBSP2 plEX-B vBSP-2 no plEX-B-vBSP3 plEX-BvBSP-3 no plEX-C-vBSP1 plEX-C vBSP-1 no plEX-C-vBSP2 plEX-C vBSP-2 noplEX-C-vBSP3 plEX-C vBSP-3 no plUS-vBSP1 plUS vBSP-1 no plUS-vBSP2 plUSvBSP-2 no plUS-vBSP3 plUS vBSP-3 no pXC-Add-eBSP pXC-Add EBSP yespS-Add-eBSP pS-Add EBSP yes

All clones were generated in E. coli and subsequently transformed incarrier bacteria.

It was then further examined whether each of the BSP fragments can beexpressed, secreted and functionally anchored onto the bacterial host.The “functional anchoring” of the BSP fragments with respect to theplanned application of the vaccine bacteria required that the BSPfragments were expressed in and translocated out of the carrierbacteria, and finally covalently anchored on the surface of thebacteria. The anchored BSP fragments contained inter alia the BSP tumorepitope, which was recognized by the tumor epitope specific antiserum.By this step it was assured that the BSP tumor epitope was actuallypresented on the surface of the carrier bacteria and that it couldinduce a tumor epitope specific immune response when vaccine bacteriawere administered for active immunization.

In order to achieve a functional anchoring of the BSP fragments, thegenerated BSP anchoring constructs were transformed into carrierbacteria and the respective BSP fragments anchored on the bacteria. Thedetection of a functional anchoring was carried out in the flow throughcytometer with a polyclonal antiserum “Anti-Human Bone Sialoprotein(amino acids 108-122) Antibody”, which is commercially available fromImmundiagnostik AG, Bensheim (Cat.-No. A4219.2, Lot H3150503). Thispolyclonal antiserum detected the tumor epitope of BSP. A positiveresult with this antiserum therefore showed the “functional anchoring”of each of the BSP fragments as heterologous Listeria surface antigensfor active immunization.

Preliminary works showed that the following BSP fragments werefunctionally expressed in bacteria and also in Listeria. Additionally,they were secreted out of the carrier bacteria and functionally anchoredon them. Hence, selected fragments of hBSP were coupled onto L.monocytogenes and the bacteria were tested for whether they induced animmune reaction against the cloned BSP fragment, the tumorgenic BSPepitope and finally against tumor cells. For this, at first, signalsequences were identified, which efficiently secreted the identified BSPfragments out of the bacteria, but only in such a way that the BSPremained anchored on the Listeria. The anchoring efficiency of theselected BSP fragments should be above 40%—if tested with antibodiesagainst these immune tags. Further, conditions were identified, underwhich the selected BSP fragments would remain anchored stably andreproducibly on the bacteria. Finally, the conditions for propagationand deactivation of the BSP bacteria for immunizations were determined.In total, the anchoring efficiency was ≧40%. However, the professionalwill have to determine the optimum breeding conditions and the bestsuited anchoring sequences for each bacterial strain in the usual way.More specifically, the following fragments of human BSP were anchored onthe surface of carrier bacteria. The anchoring efficiencies are shown inTable 3.

TABLE 3 Anchoring efficiencies BSP Fragment Vector Anchoring efficiencyvBSP-1 plEX-B >13% vBSP-2 plUS >56% vBSP-2 plEX-C >47% vBSP-3 plUS >58%vBSP-3 plEX-A >68% vBSP-4 plUS >61% vBSP-4 plEX-A >46% eBSP pS-Add >71%

FIGS. 3A-G show the results obtained with these experimentsschematically and in direct comparison with respect to fragment,anchoring vector, and breeding conditions. In order to anchor BSPfragments on the surface of carrier bacteria, two different vectorsystems were used. While the pIEX vector systems were designed for theanchoring of heterologous proteins on carrier bacteria during breedingin a fully synthetic culture medium, the pIUS vectors were suited forthe anchoring of proteins during the breeding of the carrier bacteria incomplex media.

BSP fragment vBSP-1 was anchored onto carrier bacteria most efficientlyby the secretion signal of vector system pIEX-B. The anchoringefficiency of vBSP-1 was comparatively low with 13.86% (see FIG. 3B).This is probably due to the fact that vBSP-1 is produced in the carrierbacteria only with comparatively low expression rates. Studies in otherlaboratories have shown that in most cases full length BSP (˜vBSP-1) isnot produced at all in prokaryotic expression systems. Because fulllength BSP (vBSP-1) had been anchored on the carrier bacteria in thesystem used, it can be assumed that its anchoring on the surface ofbacteria has a stabilizing effect on the full length BSP. Subsequently,BSP fragments vBSP-1, -2, -3 and -4 were functionally expressed incarrier bacteria, secreted out of the carrier bacteria and alsofunctionally anchored on the surface of carrier bacteria. Finally, thetumor epitope eBSP was expressed as fusion protein in carrier bacteria,secreted and functionally anchored. The functional anchoring was proveninter alia by FACS and monoclonal antibodies, which recognize theimmunological tag (myc-tag) fused to the C-terminal end of the BSPfragments.

Dependent on the specific biochemical properties of the protein to beanchored, different signal sequences are required for obtaining optimalsecretion and anchoring of heterologous proteins on the carrierbacteria. The inventors therefore analyzed in a test series differentsignal sequences for their ability to secret and anchor the BSPfragments.

In subsequent tests hBSP fragments vBSP-3 and eBSP were primarilystudied. The structure and position of these fragments in human BSP areshown in FIG. 2. These two BSP determinants are particularlyadvantageous since vBSP-3 contains along with the tumor epitope and twoC-terminal tyrosine-rich regions further the RGD motif. A role in theinduction of apoptosis has been attributed to the RGD motif and immunereactivities against the RGD motif, respectively. Apart from the actualtumor epitope no further BSP structures were anchored as an antigen wheneBSP or multimers of eBSP were used. This reduces the risk that theimmunization leads to reactivities against healthy cells which expressnative BSP.

Example 3*

Anchoring of hBSP FragmentsAnchoring of vBSP-3

We determined the conditions for a stable and reproducible anchoring ofthe vBSP-3 fragment. For this we tested the following constructs fortheir ability to provide a functional anchoring of the vBSP-3 fragmenton the carrier bacteria. The functional anchoring was tested byflow-through cytometry using polyclonal antibodies against amino acids108-122 of human bone sialoprotein (Immundiagnostik AG, Bensheim, DE:Cat-No. A4219.2, Lot H3150503). These polyclonal antibodies recognize anepitope on BSP, which is only present on BSP from tumor cells(heretoforth referred to as tumor epitope). A positive result with theseantibodies represents a functional BSP fragment as an antigen for anactive immunization against tumorgenic BSP.

TABLE 4 Anchoring constructs Anchoring construct Anchoring vector BSPfragment plEX-A-vBSP3 plEX-A vBSP-3 plEX-B-vBSP3 plEX-B vBSP-3plEX-C-vBSP3 plEX-C vBSP-3 plUS-vBSP3 plUS vBSP-3

The best results were got with the anchoring constructs pIEX-A-vBSP-3and pIUS-vBSP-3 (see FIGS. 3D and 3F). Optimal functional anchoring ofvBSP-3 was achieved by varying the breeding and anchoring conditionsboth by cultivation in BHI medium (Brain-Heart-Medium) as well as in asynthetic minimal medium (FIGS. 4 and 5). The achieved anchoringefficiency of greater than 52% for cultivation in BHI and minimal mediamust be considered excellent (FIGS. 4A and B). Hence, vBSP-3 bacteriacould be grown in BHI medium as well as in minimal media. The BHI-mediumgave the advantages of fast growth and high bacterial density in themedium so that a high number of vaccine doses can be produced within ashort period. On the other hand, vBSP-3 bacteria grew slower and lessdense in minimal medium. However, there is no danger of contamination ofthe vaccines with BSE or TSE when the cultivation is done in a syntheticminimal medium. This can be a decisive advantage in the future use ofvBSP-3 bacteria in humans.

Example 4

Deactivation of the Recombinant Listeria while Preserving the Anchoringof hBSP Fragments

Bacteria which carry recombinant proteins anchored to their surfaces aregenetically modified organisms (GMO). The immunization using GMOs, evenin animal models, require very extensive safety precautions. On theother hand, deactivated vaccine bacteria are not GMOs any more and maytherefore be treated like conventional vaccines.

Therefore, conditions were identified, with which the carrier bacteriamay be safely deactivated, but where at the same time vBSP-3 remainssafely anchored on the bacterial surfaces. FIG. 5 shows the anchoringefficiency of vBSP-3 on deactivated vaccine bacteria when grown in BHImedium (FIG. 5A) and minimal medium (FIG. 5B). As can be derived fromFIG. 5, the carrier bacteria could be safely deactivated by the additionof formaldehyde while the vBSP-3 remained functionally anchored on thesurfaces of the killed bacteria and was recognized by rabbit antibodiesspecific for tumor epitope. The anchoring efficiency of the vBSP-3 ondeactivated carrier bacteria was approximately 64% when grown in BHImedium and about 41% when grown in minimal medium (see FIG. 5), which isoutstanding for such types of epitopes.

Example 5

Anchoring of the BSP Fragments vBSP-2 and vBSP-4

The vBSP-3 fragment contains in addition to a glutamic acid rich regionthe two tyrosine-rich regions at the C-terminus and the RGD motif ofBSP. In comparison thereto the BSP fragments vBSP-2 and vBSP-4 lackedthe tyrosine-rich regions and the RGD motif (see FIG. 2). For optionallycharacterizing the relevance of respective functional regions of the BSPon the immune response of the vaccinated individual, vBSP2 and vBSP4vaccine bacteria were generated.

FIG. 6 shows the anchoring efficiency of BSP fragments vBSP-2 (A and C)and vBSP-4 (B and D) on carrier bacteria prior and post deactivationusing optimal experimental conditions. FIG. 6 shows that deactivatedvaccine bacteria could be produced which possessed anchoringefficiencies of close to 65% (vBSP-2 vaccination bacteria) and 68%(vBSP-4 vaccination bacteria), respectively. Thus the produced vBSP-2and vBSP-4 vaccine bacteria could well be used in immunizationexperiments.

Example 6 Anchoring of the BSP Tumor Epitope on Listeria

The initially produced anchoring constructs for the eBSP tumor epitopepossessed only anchoring efficiencies of about 6% to 7%, as determinedwith antibodies against eBSP (FIG. 7B). The low anchoring efficiencyhowever was not the result of a bad anchoring efficiency of the fusionprotein with eBSP on the bacteria. The fusion protein possessed ananchoring efficiency of nearly 70%, as determined using an antibodyagainst the carrier protein. As the BSP tumor epitope had been anchoredby way of a fusion protein with a carrier protein on the bacteria, theBSP tumor epitope must be present on the bacteria in the same proportionas the carrier protein and the anchoring efficiencies of the carrierprotein and the BSP tumor epitope must be identical. Notwithstanding,the measured anchoring efficiency of the carrier protein was close to70%, and the anchoring efficiency of the BSP tumor epitope was justbelow 7% (FIG. 7A). It is probable that the affinity of the tumorepitope BSP specific antibody was significantly lower than the affinityof the monoclonal antibody for the carrier molecule, or that the tumorepitope was shielded by the cell membrane due to its reduced size andtherefore not recognized by the antibody. It is also possible that thetumor epitope was shielded by the carrier protein. There is always achance that a singular small epitope (such as the BSP tumor epitope) isnot fully accessible. In that case the immunogenicity of suchvaccination bacteria against the tumor epitope would be low. In order toimprove the probability of a successful vaccination using eBSPvaccination bacteria, the tumor epitope was anchored onto the carrierbacteria as multimers.

Example 7 Generation of Constructs for the Anchoring of Multimeric BSPTumor Epitopes

We generated anchoring constructs that anchor the eBSP tumor epitope oneto six-fold on the surfaces of carrier bacteria (FIG. 8). A 7 amino acidspacer (-SGGGGSA-) (SEQ ID NO:8) was cloned between every tumor epitopeto ensure free flexibility of movement for each BSP tumor epitope. Acomparative analysis of the anchoring of multimeric BSP epitopes forcultures in BHI medium (FIG. 9) and minimal medium (FIG. 10),respectively, showed that the measurable anchoring efficiency ofmultimeric BSP epitopes increases between the monomer to the pentamerfrom below 10% to above 40%, but that it was slightly reduced in thecase of the anchoring of the eBSP hexamer. On the other hand, the meanfluorescence of the vaccine bacteria increased further in the case ofthe anchoring of 6x-eBSP tumor epitope, at least when grown inBHI-medium. This suggests that in the case of the eBSP tumor epitopehexamer fewer cultured bacteria carried tumor epitopes anchored to theirsurface, but that a higher number of tumor epitopes were anchored oneach bacterium compared with vaccine bacteria carrying 4x and 5xmultimeric eBSP tumor epitopes. Animal experiments will show which ofthe vaccine bacteria carrying 4x-, 5x- or 6x-multimeric eBSP have thehighest immunogenicity.

Repetitive DNA regions of 100% sequence homology are often unstable.Even though the generated constructs pIUS-mBSP1x-6x contained repetitiveDNA regions, no instability was observed for the anchoring constructsduring the experiments. Thus, stable vaccine bacteria could be producedwhich had a measured anchoring efficiency of more than 40% (see FIG. 9).The vaccine bacteria which carried the BSP tumor epitope as a 3-fold,4-fold, 5-fold or 6-fold multimer anchored to their surface displayedeven after deactivation a measurably high anchoring efficiency of above40% (FIG. 11). The anchoring rates were generally slightly higher forcultures grown in BHI medium than for cultures grown in minimal medium.Notwithstanding, vaccine bacteria with multimeric BSP tumor epitopescould be obtained both in BHI medium (FIGS. 11, C) as well as in minimalmedium (FIGS. 11, B). Thus vaccine bacteria could be provided whichcarried anchored to their surfaces BSP fragments or multimers of thetumor epitope. Table 5 below shows that anchoring rates of sometimeswell above 40% were achieved for all vaccine bacteria.

TABLE 5 Anchoring fragments and efficiencies ANCHORING EFFICIENCY BSPBEFORE AFTER FRAGMENT DEACTIVATION DEACTIVATION vBSP-2 58.4% 65.0%vBSP-3 52.3% 63.9% vBSP-4 57.8% 67.8% vBSP-5x 42.5% 40.1% vBSP-6x 34.2%46.5%

Thus, there is provided vaccine bacteria for vaccination which carry BSPfragments (vBSP-2, vBSP-3 or vBSP-4) or a multimer of the BSP tumorepitope in high amounts anchored to their surface, and which thereforetrigger an immune reaction against BSP.

Example 8 Therapeutic Activity

In order to obtain a selection for the antigens or the antibodies whichreact as well with BSP which is in a complex with factor H, factor H orBSP isolated from bones or recombinant BSP was covalently conjugatedwith cyanogen bromide-activated Sepharose 4B and than a sufficientamount of BSP and factor H, respectively, applied on the column andbound so that all the ligands in the matrix were complexed to a partner.Then, the IgG fraction from serum of immunized animals was given on thataffinity column and the antibody fraction obtained which hadspecifically bound to the free epitope in the complex of BSP and factorH. These experiments showed that particularly the 5-fold multimer vBSP5xof the eBSP epitope on vaccine bacteria elicited in rabbit extremelyhigh antibody titers where the so produced antibodies bound to human BSPwhen in complex with complement factor H (CFH). These antibodies arebeing tested for their therapeutic activity in animal trials (nude ratswith human osteotropic tumor cells) as described in WO 2002/100899.

BSP vaccine bacteria were produced with the clone pIUSind-mBSP5x for theimmunization of rabbits. The plasmid pIUSind-mBSP5x was transformed inListeria cells. The successful transformation was verified by preparingthe plasmid DNAs from transformed Listeria clones and restrictionanalysis of the plasmid DNAs. Listeria pIUSind-mBSP5x bacteria weregrown in a 5 ml culture in a suitable medium. The anchoring of the tumorepitopes took place during cultivation of the bacteria. The BSP vaccinebacteria were collected by centrifugation and deactivated by the addingof formaldehyde (up to 1% final concentration) over 24 h at roomtemperature. The quality of the BSP vaccine bacteria was assured bycharacterization of the deactivated vaccine bacteria in a flow throughcytometer. The measurement of the deactivated BSP vaccine bacteria inFACS showed that the deactivation of the vaccine bacteria had nonegative impact on the anchoring of the BSP tumor epitopes on theListeria. Listeria pIUSind-mBSP5x and Listeria pIEx-A-vBSP3 (DSM 18306and DSM 18306) have been deposited at the DSMZ (German National ResourceCentre fur Biological Material, Braunschweig) as examples.

The shuttle vector was transformed into weakly pathogenic Gram-positiveListeria. In the case of L. monocytogenes, the path into the cell ofhumans or animals is well defined. Factors such as PrfA (positiveregulator of virulence), ActA (actin nucleating protein), PlcA(phosphatidylinositol-specific phospholipase), PlcB(phosphatidyl-choline-specific phospholipase), Hly (Listeriolysin), Mpl(metalloprotease) are required for the full pathogenicity of theListeria, and may be specifically switched off for a reduction of thepathogenicity. The specificity between pathogen and host cell isimparted, among others, by internalins InlA and InlB. L. monocytogenesesmay infect endothelium cells, epithelium cells, fibroblasts andhepatocytes as well as neutrophilic granulocytes, macrophages,lymphocytes and other white blood cells. After adhesion to the cellsurface, L. monocytogenes is introduced into the cells by endocytosis.With the support of Listeriolysin (Hly), it then destroys the endosomemembrane, is released into the cytosol of the host cell, proliferatesthere while producing the cloned fragment and other proteins, andfinally infests neighboring cells. In this process, the transgenic hBSPpeptide and the cloned antigenic determinant, respectively isposttranslationally modified (O- and N-glycosylated, sulphated,phosphorylated etc.) by the host cell as well as secreted and coupledonto the Listeria bacteria. It may then elicit an autoimmune reactionagainst BSP, and especially against certain fragments or epitopes ofhuman BSP, including potential post-translational modifications. If thecarrier bacteria were grown in an artificial environment and deactivatedbefore immunization, for example by treatment with formaldehyde oraddition of an antibiotic, the antigenic determinant expressed by thecarrier bacterium was present on the surface of the carrier bacteria. Inthat case, the recombinant immune tag does not possess anypost-translational modifications.

SUMMARY

The invention provides a pharmaceutical composition for the productionof antibodies against human bone sialoprotein (hBSP), which bindsspecific epitopes on human BSP from tumor cells. The pharmaceuticalcomposition was particular optimized for the induction of antibodies,which recognize tumorgenic hBSP in serum even when in a complex withcomplement factor H. Further, the invention includes micro-organismswith a vector system for the induction of a specific somatictransgenicity in a host, especially in a human patient. The inventivecomposition particularly includes a vaccine for the induction ofantibodies against bone proteins, which are produced by osteotropictumor cells and needed for metastases into bones. The fact thatproliferating tumor cells can no longer properly post-translationallyprocess these originally strongly glycosylated proteins is indirectlyexploited and used for the production of endogenous antibodies againsttumorgenic proteins, which themselves are endogenous. Further, a methodfor vaccination and treatment of osteotropic tumors is provided whichincludes the use of the above methods, vaccines and active substances.

1-31. (canceled)
 32. A therapeutic composition for treatment andprophylaxis of bone tumors and metastases that preferentially settle inbone tissues comprising as an active ingredient dead or weaklypathogenic micro-organisms into which has been introduced at least onegene encoding a peptidic antigen of a human bone matrix protein andwhich express and secrete one or more antigens of the human bone matrixprotein and anchor them on their cell surfaces, wherein the expressedand surface-anchored antigens of the human bone matrix protein areselected to differ in their antigenic determinants from thecorresponding determinants of the endogenous human bone matrix proteinsof normal osteoblasts in at least one structural feature so that animmune reaction against the modified bone matrix protein is inducedafter administration.
 33. The therapeutic composition of claim 32,wherein the human bone matrix protein is an extracellular bone matrixprotein chosen from bone sialoprotein (BSP), osteopontin (OPN), andosteonectin (ON).
 34. The therapeutic composition of claim 32, whereinthe expressed and surface-anchored antigens of the extracellular bonematrix protein possess structural features of a bone sialoprotein thatis expressed by the osteotropic cells of a primary tumor.
 35. Thetherapeutic composition of claim 32, wherein the micro-organism ischosen from bacteria, viruses and monads.
 36. The therapeuticcomposition of claim 32, wherein the micro-organism is a Gram-positivebacterium.
 37. The therapeutic composition of claim 36, wherein themicro-organism is of the genus Listeria.
 38. The therapeutic compositionof claim 35, wherein the bacterium is chosen from the genera Aeromonas,Bartonella, Bruceila, Bacilli, Bacillus subtilis, Lactobacilli,Pseudomonades, Staphylococci, Yersinia, Campylobacter, Clostridia,Enterobacteriaceae, Legionella, Mycobacterium, Rhenibacterium,Rhodococcus, Escherichia, Shigella, Salmonella and bacteria, which areviable in a eukaryotic host organism.
 39. The therapeutic composition ofclaim 32, wherein the expressed and surface-anchored antigens of bonesialoprotein are specific for a human bone sialoprotein from tumorcells.
 40. The therapeutic composition of claim 32, wherein themicro-organism carries anchored to its surface underglycosylated humanbone sialoprotein antigen or fragments thereof.
 41. The therapeuticcomposition of claim 32, wherein the expressed and surface-anchoredantigen comprises an epitope of human bone sialoprotein from tumor cellswhich is free for specific binding by an antibody when the bonesialoprotein is in a complex with complement factor H.
 42. Thetherapeutic composition of claim 41, wherein the expressed andsurface-anchored bone sialoprotein antigen comprises one or more copiesat least one of the following amino acid sequences: YTGLAAIQLPKKAGD SEQ.ID NO. 5 TGLAA SEQ. ID NO. 3 YTGLAA SEQ. ID NO. 4 YESENGEPRGDNYRAYEDSEQ. ID NO. 6 LKRFPVQGG SEQ. ID NO. 7 EDATPGTGYTGLAAIQLPKKAG SEQ. ID NO.10


43. The therapeutic composition of claim 32, comprising as an activeingredient a protein comprising an antigenic determinant that isspecifically present on bone sialoprotein from tumor cells.
 44. Thetherapeutic composition of claim 43, wherein the active ingredientcomprises an antigenic determinant of the bone sialoprotein in at leasttwo or more copies.
 45. The therapeutic composition of claim 43, whereinthe protein with the antigenic determinant from bone sialoprotein iscoupled to beta-alanine.
 46. The therapeutic composition of claim 32,which is formulated as a vaccine.
 47. A method of prophylaxis andtreatment of bone tumors and metastatic tumor cells that preferentiallysettle into bone tissue comprising administering the composition ofclaim 32 to a subject to produce an immune reaction against said bonematrix proteins and cancer cells expressing said bone matrix proteins.48. The method of claim 46, wherein the tumor cells are from tumorsselected from the group consisting of tumors of the prostate, breast,lung, kidney, thyroid, circulatory system, lymphoid system,cardio-vascular system, neurological system, respiratory tract,digestive tract, endocrine system, skin, adnexa, musculoskeletal systemand the urogenital system.